2011 101 lab 2 tutor template.pdf

8/14/2019 2011 101 Lab 2 Tutor template.pdf

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BIOSCI.101 Laboratory Course

TUTOR:

DEMONSTRATORS:

This Course has six labs:

1. Enzymology

2. Gene Expression

.4. Evolution

5. Blood Glucose

6. Photosynthesis


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Reminder Lab Organisation

Have you handed in your pre-lab assignment? Things to bring to every laboratory:

Manual (having read the lab!)

Pen, marker pens and pencil

Ruler

Calculator

a ora ory oa

If you miss a lab fill out an Absence fromLaboratory form, attach a medical certificate, handit to the Course Coordinator - Mandy Harper.

Your assignment Sheet! Answer as you go & write in pen. Answers are almost alwa s somewhere in the

text in lab &/or lecture guide OR in thepresentation!

PAY ATTENTION

Write carefully and tidily.

We cant mark what we cant read.

Get your assignment sheet SIGNED OFFbefore you leave


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Exercise 2.1 (pg 2.5): RNA hydrolysis

Start with this

exercise

o n ac con ons se u e w

black screw cap)

Acid hydrolysis breaks phosphodiester linkage

Separate nucleotides by chromatography

Laboratory is in two parts

.

2. Control of Gene expression

Read each shaded box in the lab

each experiment


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The Central Dogma of molecular biology

Basically once information is converted to protein, it cannotflow back to nucleic acid

Transcription

DNA

mRNA

In the cell

information is

stored

where?

Copyright 2002 Pearson Education, Inc., publishing as Benjamin Cummings

Translation

ribosome

polypeptide

protein

Nucleic acids store andtransmit hereditary information

ribonucleic acid (RNA) and

deoxyribonucleic acid (DNA).

DNA provides direction for its own

replication.

DNA also directs RNA synthesis and,

through RNA, controls protein synthesis.


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A nucleic acid strand is a polymer of

nucleotides

Each nucleotide consists of three parts

Base + Sugar

=

uc eos e

Another name for a nucleotide is nucleoside monophosphate

There are two types of nucleotides

6-membered ring

6-membered ring

joined to a 5-

membered ring


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DNA base pairing

Base pairing:

A T

G C


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The sugars of one nucleotide are joined to thephosphate of the next nucleotide via

This creates a sugar phosphate backbonePHOSPHODIESTER BONDS

Base pairing:

A T

G C

Nucleic Acid Structure Important Terms

DenaturationDe = Un, Nature = natural state or configuration

GENTLE BREAK WEAK BONDS i.e.

HYDROGEN BONDS BETWEEN BASES

Hydrolysis

From Greek: Hydro = water, Lysis = breakbreaking using water

HARSH WILL BREAK PHOSPHODIESTER

LINKAGES


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Hydrolysis

The covalent bonds connecting monomers in a polymerare disassembled by hydrolysis

In hydrolysis as the covalent bond is broken ahydrogen atom and hydroxyl group from a split watermolecule attaches where the covalent bond used to be

DENATURATION

HYDROLYSIS


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Inheritance is based on replication of theDNA double helix

DNA is relatively stable, hence ANCIENT DNA

DNA double stranded double helix

A sing e stran e extra O group in

presence of OH radicals) can attack thephosphodiester bond

- Spontaneously at neutral pH and is called auto-catalytic-cleavage

Compared to DNA, RNA is relatively unstable

RNA single stranded & extra OH group

OH group can attack the phosphodiesterbond occurs spontaneously at neutral andalkaline H auto-catal tic-cleava e


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Precipitation and denaturation of DNA/RNA

RNA and DNA have different propertiesue o s zes an s ng e ou e-s ran e

nature. To investigate this:

2.2 Add ethanol and NaCl to DNA/RNAand observed any precipitate that forms

2.3 Place DNA and RNA in boiling waterbath for ~5 minutes and repeat

Back to the RNA hydrolysis

The covalent bonds connecting monomers in a polymerare disassembled by hydrolysis

In hydrolysis as the covalent bond is broken ahydrogen atom and hydroxyl group from a split watermolecule attaches where the covalent bond used tobe


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Chromatography and precipitations

Start precipitations exercises 2.2 and 2.3

Hydrolysis cool for 10 minutes whileyou wait load the standards on your plates then spot the plates with the Tube Asamples

ALL DONE? Pick up bacterial plates andstart reading ahead, there will be a lacoperon talk

2.1 RNA hydrolysis

phosphodiester bonds

broken

RESULT: ribonucleotides


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RNA hydrolysed to ribonucleotides

purines nucleotidesunstable, breakdown to

release bases

Four RNA hydrolysis products


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RNA hydrolysis (2.1)

After hydrolysis cool for 10 minutesw e you wa oa e s an ar s on yourplates then spot the plates with theTube A samples

Thin-layer chromatography

-


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Afer the plates have dried, place in tanks(2 rou s/tank) under su ervisionDevelop for as long as possible (> 1 hour),i.e. visualise ~4:30pm

Thin-Layer Chromatography

standards


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View in UV viewing box (basesa sor uoresce un er g


solvent will carry

different molecules

at different rate


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Chromatography tanks


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Viewing Chromatograms

Lab 2 Gene ExpressionThe Lac Operon

.

genotype of stains A, B, C

2.5 Induce -galactosidase with

lactose/IPTG & determine effect of

glucose


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B Plate

using this format.

A

B+I Plate

using this format.B

A


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lac Operon in E. coli

repressorpromoter

negative gene regulation

positive gene regulation

via cAMP receptor protein

Low glucose

mRNA

lacZ lacY lacA

mRNAcAMP

permease transacetylase

repressor

protein

lactose

(inducer)

Inactive repressor

+

lactose

-gal = enzyme

lactosegalactose glucose

Transcription of -gal

occurs:Lactose (inducer) is

present

And glucose is absent

(cAMP is high)

lac Operon in E. coli

repressorpromoter

lacZ lacY lacA

mRNAmRNA

permease transacetylase

repressor

protein

IPTG(inducer)

Inactive repressor

+

IPTG

-gal = enzyme


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2.4 Different strains of E.coli

Analysis of plates Think: in which plate is inducer present? B+I plate, no inducer on B plate

Think: what do mutations do? -galactosidase mutation:

non-functional enzyme

Operator mutation: repressor cannot bind i.e. cant switch off

What colour will strains be? - ype

white without inducer, blue with inducer

-galactosidase mutation always white

Operator mutation always blue

2.5 Induce -galactosidase

. ,to tubes as given in book (step 2)

Incubate for 20 minutes at 37C, add lysismedium & tap

Incubate for 15 minutes at 37C

Add ONPG and incubate for 5 minutes

Observe colour of each tube, note colour insome tubes may be pale

Start ASAP, can do other tasks while waitingfor incubations


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2011 101 lab 2 tutor template.pdf

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